The present invention is in the field of allergy and relates to peptides derived from the complement peptide C3a and to their use in the prevention and treatment of allergies.
Mast cells and basophils play a central role in inflammatory and immediate hypersensitivity reactions.
Clustering of the type 1 Fcxcex5 receptors (Fcxcex5RI) present in their plasma membranes initiates a coupling cascade culminating in the secretion of mediators of immediate-type allergic reactions (Schwartz, 1994). The molecular mechanism of signal transduction initiated by Fcxcex5RI clustering has been studied intensely over the past few years (Ravetch et al, 1991; Holowka et al, 1992; Benhamou et al, 1992; Beaven et al, 1993). The xcex2 and xcex3 subunits of the clustered Fcxcex5RI were found to interact with src family protein tyrosine kinases (PTK) and cause the so far earliest detected biochemical event in their cascade, i.e., their activation. The differential control of these PTK by the xcex2 and xcex3 chains of the receptor has also been described recently (Jouvin et al, 1994). The following steps downstream involve recruitment of PTK of the syk family, activation of phospholipase Cxcex3, which in turn leads to hydrolysis of phosphatidyl inositides and production of inositol triphosphate and diacylglycerol. The former product causes the transient increase in free cytosolic calcium ion concentration while the latter is involved in activation of protein kinase C (Ravetch et al, 1991; Holowka et al, 1992; Benhamou et al, 1992; Beaven et al, 1993).
Two types of mast cells differentiate from a common precursor to produce the so-called serosal (or connective tissue type) and the mucosal type mast cells. Both phenotypes express Fcxcex5RI on their cell membrane; however, they respond differently to secretagogues and inhibitors. For example, only serosal-type mast cells are triggered by cationic peptides (including the complement-derived peptides C3a and C5a; venom-peptides, e.g., mastoparan, mellitin; neuropeptides) or polyamines; mucosal mast cells are non-responsive to these stimuli (Mousli et al, 1994). In comparison to the advanced understanding of the coupling cascade initiated by Fcxcex5RI clustering, the action modes of the complement-derived peptides C3a and C5a are less well understood.
As mentioned above, clustering of the Fcxcex5RI on mast cells and basophils is known to trigger the secretory response of these effector cells central to the allergic reactions. Similarly, the anaphylatoxic activity of the complement-derived peptides C3a and C5a is also well-known, causing activation of serosal type mast cells that results in the release of histamine or serotonin and several other inflammatory mediators including proteases, lipid mediators and several cytokines (Schwartz, 1994).
Cellular events triggered in mast cells and basophils upon Fcxcex5RI clustering or by complement-derived peptides have been investigated so far only as independent physiological processes. However, in vivo, both stimuli might be exerted simultaneously.
In earlier experiments carried out by the inventors of the present application, both the secretory response and biochemical coupling processes initiated by Fcxcex5RI clustering of rat mucosal mast cells of the line RBL-2H3, which is non-responsive to complement-derived peptides, were investigated in the presence of a range of human C3a concentrations. It was found that C3a inhibits dose-dependently antigen-induced degranulation of the mucosal line RBL-2H3 while C5a applied in the same amounts had no effect at all. When added alone, none of the complement-peptides C3a and C5a triggered the secretory response of the rat mucosal-type cells of the line RBL-2H3 (Erdei et al, 1995).
Several steps coupling the Fcxcex5RI-mediated stimulus to the secretory response were tested in order to try and identify at which point of the cascade C3a interferes. It was found that neither antigen binding to IgE-sensitized cells nor the reaction of Fcxcex5RI with IgE were influenced by the complement peptide C3a. However, the tested intracellular events were strongly and dose-dependently inhibited by C3a, i.e., tyrosine phosphorylation of several cellular proteins, the activity of PLCxcex3, resulting in the inhibition of antigen-induced hydrolysis of phosphatidyl inositides, and elevation of intracellular free Ca2+. RBL-2H3 cells proved to be unresponsive to C3a in all these tests.
The fragment C3a, also called anaphylatoxin, is not suitable for use as an anti-allergic drug because it is anaphylatoxic to serosal mast cells, i.e., it is capable of causing mediator release from this type of mast cells.
It has now been found in accordance with the present invention that certain peptides comprised partially or entirely within the sequence of positions 50-77 of the complement-derived peptide C3a, and analogs thereof, are capable of inhibiting the Fcxcex5RI-induced secretory response of mucosal mast cells, without having the anaphylatoxic effect of C3a to mucosal mast cells.
Peptide C3a is a 77-mer peptide of the sequence:
Ser-Val-Gln-Leu-Thr-Glu-Lys-Arg-Met-Asp-Lys-Val-Gly-Lys-Tyr-Pro-Lys-Glu-Leu-Arg-Lys-Cys-Cys-Glu-Asp-Gly-Met-Arg-Glu-Asn-Pro-Met-Arg-Phe-Ser-Cys-Gln-Arg-Arg-Thr-Arg-Phe-Ile-Ser-Leu-Gly-Glu-Ala-Cys-Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-Ala-Arg (SEQ ID NO:1)
The present invention relates to a peptide corresponding partially or entirely to the 50-77 sequence of the complement-derived peptide C3a and to analogs thereof capable of inhibiting IgE-mediated triggering and/or the Fcxcex5RI-induced secretory response of mucosal mast cells, said peptides being selected from the sequences:
(a) X1-Cys-Asn-R1-Ile-Thr-R2-Leu-R3-R4-Gln-His-R5-R6-R7-R8-R9-R10-Gly-Leu-Ala-R11 (SEQ ID NOs:2-4);
(b) X1-Cys-Asn-R1-X4 (SEQ ID NOs:5-13);
(c) X2-Lys-Val-Phe-Leu-Asp-X3 (SEQ ID NOs:14-17; and
(d) X5-Asp-Ser-Ser-Asn-Tyr-Ile-R11 (SEQ ID NO:18) wherein
X1 is H, lower alkanoyl, Cys, Asp-Cys or Arg-Arg-Cys;
X2 is H, lower alkanoyl or Lys;
X3 is Arg or a sequence selected from
(i) Ala-Ala-Asn-R1-Ile-Thr-R2-Leu-R3-R4 (residues 7-16 of SEQ ID NO:15);
(ii) Cys-Cys-Asn-R1-Ile-Thr-R2-Leu-R3 (residues 7-15 of SEQ ID NO:16); and
(iii) Cys-Cys-Asn-R1-Ile-Thr-R2-Leu-R3-R4-Gln-His-R5-R6 (residues 7-20 of SEQ ID NO:17);
X4 is Gly, (i) Ile-Thr-R2-Leu-R3 (residues 5-9 of ID NO:6); or (ii) Ile-Thr-Arg-R11 (residues 5-8 of SEQ ID 7);
X5 is H, lower alkanoyl or Leu;
R1 is an aromatic amino acid residue;
R2 is Glu or Lys;
R3 is a positively charged amino acid residue;
R4 is Arg or Glu;
R5 is Ala or Arg;
R6 is Arg or Lys;
R7 is Ala or Asp;
R8 is Ser or His;
R9 is His or Val;
R10 is Leu, Ile, Ala or Gly; and
R11 is OH, Arg, Arg-NH2, or Agm (agmatine);
and chemical derivatives and pharmaceutically acceptable salts thereof.
The peptide of the invention has preferably at least 5, more preferably 5, 7-8 or 20-21, and at most 28, amino acid residues. According to the invention, R1 is an aromatic amino acid residue preferably selected from Phe, Tyr, His and Trp; and R3 is a positively charged amino acid residue preferably selected from Arg, D-Arg, Har (homoarginine) and Lys. Lower alkanoyl according to the invention has preferably 1-4 carbon atoms, e.g., formyl, acetyl, propanoyl and butyryl.
In one embodiment, the peptide of the invention is the peptide herein identified as peptide C3a2, a 21-mer corresponding to the 57-77 sequence of the human complement peptide C3a, or the amide thereof, of the sequence:
Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-Ala-Arg (residues 57-77 of SEQ ID NO:1)
In another embodiment, the peptide of the invention is the peptide herein identified as peptide rC3a2, a 21-mer corresponding to the 57-77 sequence of the rat complement peptide C3a, or the amide thereof, of the sequence: Cys-Asn-Tyr-Ile-Thr-Lys-Leu-Arg-Glu-Gln-His-Arg-Arg-Asp-His-Val-Leu-Gly-Leu-Ala-Arg (SEQ ID NO:19)
In a further embodiment, the peptide of the invention is the peptide herein identified as peptide C3a2-R, a 20-mer corresponding to the 57-76 sequence of the human complement peptide C3a, or the amide thereof, of the sequence:
Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-Ala (residues 57-76 of SEQ ID NO:1)
In yet a further embodiment, the peptide of the invention is the peptide herein identified as peptide C3a9, a 8-mer analog corresponding to the 55-62 sequence of the human complement peptide C3a (with Glu62 changed to Arg), or the amide thereof, of the sequence:
Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Arg (SEQ ID NO:20)
In still a further embodiment, the peptide of the invention is the peptide herein identified as peptide C3all, a 7-mer analog corresponding to the 55-61 sequence of the human complement peptide C3a (with Cys56 and Cys57 each changed to Ser and Thr61 changed to Arg), or the amide thereof, of the sequence:
Asp-Ser-Ser-Asn-Tyr-Ile-Arg (SEQ ID NO:21)
In yet another embodiment, the peptide of the invention is the peptide herein identified as peptide C3a13, a 5-mer analog corresponding to the 56-60 sequence of the human complement peptide C3a (with Ile60 changed to Gly), or the amide thereof, of the sequence:
Cys-Cys-Asn-Tyr-Gly (SEQ ID NO:22)
In still further embodiments, the peptides of the invention are the 14-mer C3a4, 20-mer C3a5, 15-mer C3a6, 9-mer C3a7, 7-mer C3a8, 10-mer C3a10, and 6-mer C3a12 and the 7-mer C3a14-P peptides, or the amides thereof, of the sequences:
C3a4: Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg (residues 51-64 of SEQ ID NO:1)
C3a5: Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg (residues 50-69 of SEQ ID NO:1)
C3a6: Lys-Val-Phe-Leu-Asp-Ala-Ala-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg (SEQ ID NO:23)
C3a7: Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg
C3a8: Lys-Val-Phe-Leu-Asp-Arg (residues 56-64 of SEQ ID NO:1)
C3a10: Arg-Arg-Cys-Cys-Asn-Tyr-Ile-Thr-Arg-Arg (SEQ ID NO:25)
C3a12: Asp-Cys-Cys-Asn-Tyr-Gly (SEQ ID NO:26)
C3a14-P: Asp-Ser-Ser-Asn-Tyr-Ile-Thr-Arg (SEQ ID NO:27)
The invention further relates to pharmaceutical compositions comprising at least one peptide of the invention, a chemical derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for the prevention and/or treatment of allergic disorders caused by IgE mediated (Type I) hypersensitivity where mucosal-type mast cells are involved such as hay fever, asthma rhinitis, some cases of urticaria, allergic conjunctivitis, and the like.
The invention further relates to a method for the prevention and/or treatment of allergic disorders caused by IgE mediated (Type I) hypersensitivity where mucosal-type mast cells are involved, which method comprises administering to an individual in need thereof an effective amount of a peptide of the invention, a chemical derivative or a pharmaceutically acceptable salt thereof.